High Pressure Freezer for Improved Ultrastructure

DESCRIPTION (provided by applicant): The users assembled in this application have a common interest in preparing small samples for transmission electron microscopy. High Pressure Freezing (HPF) allows the fastest high quality fixation of tissue samples (200 microns thickness x 2mm diameter) for high resolution electron microscopy. Preliminary studies by the investigators have shown that we can produce "life-like" views in thin section of biological events such as endocytosis and glycocalyx changes which were rarely if ever seen previously by conventional chemical immersion fixation. Similar improvements can also be carried to high resolution immunocytochemistry, where enhanced tissue preservation is highly desirable. Many of these experiments will utilize immunogold techniques following HPF, freeze substitution and embedding in low temperature resins to localize proteins or mRNA in various tissues and cell types, all of which will benefit from improved spatial resolution. The grant is based upon funded projects in which this technique offers substantial improvements over current methods for animal cells, whole tissues, yeast and bacteria. Dr. Hall will be applying this technique to study mutations of the model organism Caenorhabditis elegans affecting tissue differentiation, including cytoskeleton, organelle integrity, and synaptic structure. The Major Users will utilize the machine to characterize the mitotic spindle in Drosophila embryos (Dr. Sharp), the structure of caveoli in yeast and in knockout mice (Dr. Lisanti), the distribution of mRNAs in yeast (Dr. Singer), and podocytes in the kidney glomerulus of knockout mice (Dr. Mundel). The machine can also prepare frozen samples for cryosectioning prior to cryeEM of the frozen sample. With this technique, Dr. Singer can conduct 3D tomographic reconstruction of the yeast Iocosome and Dr. Mundel will reconstruct the podocyte slit diaphragm.